E2F reporter melanoma cells

ABSTRACT

A method of using a modified melanoma cell capable of quantification of the effects of MEK inhibitors and CDK4/6 inhibitors in a quantitative, temporal and non-invasive manner both in vitro and in vivo.

FIELD OF INVENTION

The present application is generally related to modified reporter cellsand methods of testing and/or detection of inhibitory compounds forefficacy in inhibiting E2F-mediated activity. In particular, methodsincluding an assay to assess the efficacy of a cyclin-dependent kinase(CDK) inhibitor to block E2F signaling that is non-invasive, andprovides quantitative results in a non-bias manner, that is capable ofmonitoring activity across the whole of the tumor.

BACKGROUND OF THE INVENTION

Typically, aberrant cell cycle is a hallmark of cancer. A majorchallenge in oncology treatment is to identify compounds that targetspecific tumor types in order to minimize toxicity and to maximizeefficacy to the targeted tumor.

In the oncology field, it is common to identify tumor types usingmicroscopic histopathological appearance of fixed and stained tumorsamples, and to use systems such as the tumor-node-metastasis system todetermine the clinical spread of the tumor. This, and other systems,evaluate the size of the tumor, the presence or absence of the tumor inthe lymph nodes, and the presence or absence of metastases to assign astage to the tumor. The tumor type and the stage are then frequentlyutilized to select appropriate therapy and to determine prognosis forthe patient.

E2F transcription factors control the expression of many genes and playa major role during the G1-S transition in the cell cycle. Severalassays currently exist that allow for testing of E2F inhibition;however, these assays suffer from several problems.

For example, current assays are frequently invasive, in that theyrequire significant sampling of a tumor tissue. Other assays are notquantitative, that is, they may report a yes or no answer, but areunable to appropriately quantify and measure, in a non-bias manner, theamount or percentage of block of E2F signaling. Furthermore, currentassays require that the experiment is terminated at the time of sampleretrieval and therefore cannot be utilized to test E2F activity in atemporal manner.

Accordingly, there is a need to identify new methods and materials thatare suitable for determining the efficacy of an inhibitor to block E2Fsignaling in a non-invasive manner and which is quantitative in anon-bias manner so as to properly identify and quantify the amount ofblock on a particular inhibitor.

SUMMARY OF THE INVENTION

In accordance with these and other objects, a first embodiment of aninvention disclosed herein is a transduced cancer cell comprising anEGFP-firefly luciferase fusion gene, under the control of E2Ftranscriptional response elements, wherein the cell is suitable fortesting the ability of a compound to block E2F signaling and to quantifythe level of block against a control.

An embodiment is directed to a method for determining the efficacy of acompound of interest to inhibit E2F activity comprising: applying acompound of interest to a modified cell, wherein said melanoma cellsharboring either BRAF/NRAS mutations or neither mutations involving amodified E2F reporter system to monitor the efficacy of CDK4/6inhibitor-based therapies, wherein tumor cells are transduced withtdTomato fluorescent protein and an EGFP-firefly luciferase fusion geneunder the control of E2F transcriptional response elements; determiningthe amount of tdTomato activity and determining the amount of fireflyluciferase activity in the modified cell, and comparing the amount oftdTomato to the amount of firefly luciferase expression, to quantitateE2F reporter activity to tumor size, wherein tdTomato measures tumorsize and firefly luciferase measures pathway activity in the tumor.

A further embodiment is directed to a high-throughput in vitro screeningmethod for determining inhibition of E2F comprising: loading reportercells comprising an E2F response element-EGFP-firefly luciferaseconstruct onto a screening plate, mixing at least one compound ofinterest in a test vehicle, administering to at least one cell on saidscreening plate the compound of interest, administering to at least asecond cell, the test vehicle, incubating said cells for a predeterminedamount of time, comparing the amount of firefly luciferase activity tothe level of actin/tdTomato activity between the compound of interestand of the test vehicle in the incubated cells, and quantifying thelevel of block for the cells treated with the compound of interest, bynormalizing firefly luciferase activity to tdTomato activity/actinlevels.

A further embodiment is directed to a kit for testing and determiningefficacy of a compound to inhibit E2F activity comprising: (1) E2Fresponse elements-EGFP-Firefly luciferase plasmid/lentivirus, and (2)tdTomato fluorescent protein plasmid/lentivirus, wherein, the fireflyluciferase and tdTomato fluorescent protein are transduced into a cancercell to generate a reporter cell line of interest.

A further embodiment is directed to a kit for transducing a cancerouscell, comprising (1), E2F response elements-EGFP-firefly luciferaseconstruct and (2), tdTomato fluorescent protein, wherein the E2Fconstruct and tdTomato protein can be transduced into a cancer cell,wherein said cell can be suitably used in in vitro or in vivo screeningto test a compound for its ability to inhibit the E2F pathway and toquantify the level of block of the same.

A further embodiment is directed to a method to monitor the efficacy ofa compound on the E2F Pathway, including CDK4/6 based inhibitors,utilizing modified cells involving an E2F reporter system. Cancer cellsare transduced with tdTomato fluorescent protein to specifically measuretumor size and an EGFP-firefly luciferase fusion gene under the controlof E2F transcriptional response elements to measure pathway activity.Cells are then appropriately treated with a compound of interest and theamount of tdTomato can be compared to the firefly luciferase activitytherein. By comparing the amount of tdTomato to the amount of fireflyluciferase expression, the results of inhibition can be quantified.

A further embodiment is a method for screening novel compounds forinhibitory properties on E2F activity comprising applying a compound ofinterest to modified E2F reporter cells, comparing the amount oftdTomato expression to the amount of firefly luciferase activity in thecells and quantifying the results of the inhibition of the compound suchas a CDK4/6 inhibitor.

A further method is a method for testing inhibitory compounds withregard to efficacy for inhibition of the E2F pathway throughestablishment of a cell-based E2F reporter system that provides forquantitative analysis of pathway inhibition in vivo and in vitro. Themethod comprising establishing a cell-based E2F reporter system asdescribed herein, wherein the method comprises determining the efficacyof an inhibitor to block E2F signaling in melanoma cells in anon-invasive manner, and provides that the amount of inhibition can bequantified by comparing the amount of expressed firefly luciferase ascompared to the amount of expressed tdTomato.

A further embodiment is directed to a high-throughput in vitro screeningmethod, comprising: loading reporter cells comprising an E2F responseelement-EGFP-firefly luciferase construct onto a screening plate, mixingat least one compound of interest in a test vehicle, administering to atleast one cell on said screening plate the compound of interest, andadministering to at least a second cell, the test vehicle, incubatingsaid cells for a predetermined amount of time, after incubating,comparing the amount of firefly luciferase activity to the level ofactin/tdTomato activity between the compound of interest and of the testvehicle, and quantifying the level of block for the cells treated withthe compound of interest, by normalizing firefly luciferase activity totdTomato activity/actin levels.

A further embodiment is directed to a high-throughput screening kitcomprising a plate comprising a plurality of wells, and disposed withinsaid wells are a predetermined number of transduced cells comprising anE2F response element-EGFP-firefly luciferase construct, wherein thecells within a well can be administered a compound of interest, whereinthe compound and cells are incubated for a predetermined amount of time.Intracellular luciferase is analyzed by lysing the cells and aluciferase substrate, luciferin is added to the cell lysates andluminescence signal can be measured using a luminometer. tdTomatofluorescent protein can be measured using fluorometer or actin levelsmeasured using a western blot. Pathway activity, which identifies thepercent of block the compound of interest towards the E2F signalingpathway can then be quantified by normalizing firefly luciferaseactivity to tdTomato activity/actin levels.

A further embodiment comprises a system for determining whether acompound is efficacious for inhibiting CDK4/6 in a melanoma cellcomprising:

-   -   a. a transduced cell with tdTomato fluorescent protein and        EGFP-firefly luciferase fusion gene under the control of E2F        transcriptional response element;    -   b. applying a compound for inhibition of the CDK4/6 pathway; and    -   c. comparing the fluorescence of tdTomato to firefly luciferase        activity to quantify the amount of inhibition of the pathway.

A further embodiment is directed to a method for determining block ofthe E2F pathway comprising; generating a cell line comprising (1), E2Fresponse elements-EGFP-firefly luciferase construct and (2), tdTomatofluorescent protein; implanting said cells subcutaneously orintradermally into an immunodeficient nude mouse, treating said mousewith compound of interest for a certain amount of time, measuring theamount of firefly luciferase by intraperitoneal injection of luciferinin the mouse and subsequent utilization of an IVIS in vivo imagingsystem. In parallel, fluorescence intensity of tdTomato within the tumoris imaged. By normalizing the firefly luciferase activity to tdTomatoactivity or tumor volume, the in vivo method can be used to test anddetermine the efficacy of a compound of interest for block of the E2Fpathway in a temporal manner, and also to monitor for resistance of thedrug. Resistance to the compound of interest can be tested by monitoringfirefly luciferase activity and tdTomato activity in a temporal mannerand looking for an increase in the value of firefly luciferase activitynormalized to tdTomato fluorescence. Typically, resistance is evidentthrough a significant increase in the value of firefly luciferaseactivity normalized to tdTomato fluorescence, such as a greater than 10%increase in a day, but may include a 25%, 50%, 100% or more increaseover a period of between about one and seven days.

A method for determining the level of block of a compound of interestcomprising; performing a first in vitro screen of said compound,comprising administering said compound to a reporter cell model,incubating said cell and compound, measuring luciferase and tdTomatofluorescence in said cell after said incubation period and determiningthe level of block; performing a second screen, comprising of an in vivoscreen of said compound, comprising implanting said cells subcutaneouslyor intradermally into an immunodeficient nude mouse, treating said mousewith compound of interest for a certain amount of time, measuring theamount of firefly luciferase and tdTomato fluorescence by imaging themouse utilizing an IVIS in vivo imaging system and comparing theintensity of firefly luciferase to the intensity of tdTomatofluorescence.

The embodiments described above can be suitably utilized with a cancercell. In certain preferred embodiments, it is advantageous to utilize amelanoma cell as the cancer cell, however, other embodiments maysuitably use a cancer cell including, a non-limiting list including acell from a cancer of breast, mouth, lung, pancreas, esophageal, uveal,colon, prostate, or other cancer of interest. The cancer cells can beadvantageously modified with the reporter mechanism as described herein,whereby compounds of interest can be suitably tested against said cellsfor efficacy of said compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an overview of the CDK4/6 pathway in melanoma.

FIG. 2 depicts an overview of the effects of CDK4/6 inhibitor(Palbociclib) in vitro.

FIGS. 3A-3E depict cytostatic effects of palbociclib in melanoma.

FIGS. 4A-4C depict enhanced effects of combined MEK and CDK4/6inhibition in BRAF and NRAS mutant lines in vivo.

FIGS. 5A-5G depict sensitivity to combined CDK4/6 and MEK inhibitionassociated with survivin depletion in sensitive cells.

FIGS. 6A-6E depict survivin as essential for the survival of melanomacells.

FIGS. 7A-7H depict an embodiment of the E2F reporter model.

FIG. 8 depict an in vitro screen with silencing RNA to CDK4 and CDK6utilizing 1205Lu E2F reporter cells.

FIGS. 9A-9C depicts tumor regrowth following MEK and CDK4/6 inhibitorwithdrawal. E2F reactivation precedes tumor regrowth followingwithdrawal.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The embodiments of the invention and the various features and advantagesthereto are more fully explained with references to the non-limitingembodiments and examples that are described and set forth in thefollowing descriptions of those examples. Descriptions of well-knowncomponents and techniques may be omitted to avoid obscuring theinvention. The examples used herein are intended merely to facilitate anunderstanding of ways in which the invention may be practiced and tofurther enable those skilled in the art to practice the invention.Accordingly, the examples and embodiments set forth herein should not beconstrued as limiting the scope of the invention, which is defined bythe appended claims.

As used herein, terms such as “a,” “an,” and “the” include singular andplural referents unless the context clearly demands otherwise.

As used here, the term “about” refers to within 10% of a stated number.

Aberrant cell cycle progression is a hallmark feature of cancer. Thecell cycle consists of distinct phases: G0 (quiescence), G1 (pre-DNAsynthesis), S (DNA synthesis), G2 (pre-division) and M (cell division)and is tightly regulated by a network of cyclin dependent kinases(CDKs), cyclins and CDK inhibitors (CDKI). Positive interaction betweenCDKs and cyclins drives cell cycle progression, whereas CDKIs act as abrake by negatively regulating CDK activity. Commitment to the cellcycle occurs in G1 phase and involves CDK4/6 in association with D-typecyclins contributing to the inactivation of the tumor suppressor,retinoblastoma (RB). This uncouples RB from E2F transcription factors,which allows E2F to drive the transcription of E2F-regulated genes.Although interphase CDKs are targetable, early generation CDK inhibitors(e.g. flavopiridol) were non-selective and showed limited therapeuticvalue in melanoma patients.

Inhibitors of cell cycle components, CDK4/6, are currently being testedin clinic with significant advances being made in breast cancerresearch. The response of cancer cells to CDK4/6 inhibitors relies onthe presence of tumor suppressor RB that binds to and inhibits E2F fromtranscribing its target genes. It is expected based on studies thateffective inhibition of E2F will be correlated with positive clinicalresponses. The recent FDA approval of an orally available, highlyselective inhibitor of CDK4/6, palbociclib (IBRANCE/PD-0332991) inpost-menopausal estrogen receptor (ER)-positive/HER2-negative breastcancer has rekindled interest in targeting cell cycle progression incancer. Palbociclib also showed clinical activity in mantle celllymphoma with oncogenic t(11;14)(q13;q32) translocation resulting in theaberrant expression of cyclin D1.

In melanoma, multiple mechanisms drive aberrant progression through thecell cycle leading to uncontrolled proliferation; thus, providing arationale for therapeutically targeting CDK4/6. Mutations in BRAF (˜50%frequency) and in NRAS (15-20%) activate the MEK-ERK1/2 pathway, whichupregulates cyclin D1. Inactivation of RB1 also occurs through CDK4mutation, loss of functional CDKI proteins such as p16INK4A and p14ARF,and, to a lesser degree, loss of RB1 itself. In view of FIG. 1, anoverview of the CDK4/6 pathway in melanoma is depicted. FIG. 1identifies the pathway beginning with RAS, RAF, MEK, ERK, Cyclin D, CDK4/6, wherein activated CDK4/6 can phosphorylate tumor suppressor, RBleading to the inactivation of RB.

This knowledge has led to studies analyzing the effects of targetingCDK4/6 in melanoma (FIG. 2). Indeed, FIG. 2, depicts a flow-chart ofpalbociclib, a CDK4/6 selective inhibitor, wherein Palbociclib inhibitsG1-S progression in vitro and provides for anti-tumor activity inxenografts. In vitro studies show that loss of functional p16INK4Athrough deletion, hyper methylation or mutation in melanoma cellscorrelated with palbociclib sensitivity. Mutant NRAS extinction in aninducible NRAS genetically engineered mouse (GEM) model decreased cellcycle progression via effects on the expression of CDK4 and increasedapoptosis via inhibition of the MEK-ERK1/2 pathway. Furthermore,simultaneous targeting of MEK and CDK4 inhibited tumor growth of mutantNRAS SB-2 cell xenografts.

Given the promising development of cell cycle intervention in breastcancer, mantle cell lymphoma, and melanoma, it is expected thatsignificant development of old or new compounds will progress, whereinit will be necessary to determine a response to CDK4/6 inhibitors aloneand in combination with other clinically relevant targeted agents fornot just these cancers, but any number of cancer types. This isimportant to identify subgroups that will likely benefit from CDK4/6inhibitors and to assist in patient selection in clinical studies.Therefore, a reporter system that can provide for in vitro and/or invivo screening protocols providing a new opportunity to test andquantify efficacy of compounds of interest, and also includes theopportunity to determine whether certain cells have resistance to thecompounds of interest, is of great value.

Applicants have created an E2F-dependent luciferase reporter cell lineto enable temporal quantification of the effects of CDK4/6 inhibitorbased treatments in a quantitative, non-invasive, and tumor-selectivemanner. In this reporter system, hyper-phosphorylation of RB1 leads tothe uncoupling of E2F and subsequent E2F-mediated induction of fireflyluciferase activity. 1205Lu cells were chosen based on their ability toform tumors in vivo and their utility in a previously developed ERK1/2reporter system. However, no current method exists to quantify E2Factivity in melanoma cells in real-time known methods and current assayshave the ability to detect inhibition, but there is no way toquantifying the amount of inhibition in such cells by the particularinhibiting agent. Therefore, when comparing the efficacy of one agentover another, there is a need for the ability to quantify the results toappropriately determine the efficacy of a compound.

The cells described herein, and the methods of using the same, providefor the ability to quantify and compare the efficacy of compounds thatinhibit the E2F pathway, including inhibition of CDK4/6 activity.Modified cells include a reporter system comprising E2F responseelements-EGFP-firefly luciferase construct and a tdTomato fluorescentprotein that express a reporter that allows compounds to be screenedagainst a control, to test for compounds that alter the activity of theE2F pathway. The reporter system, unlike currently known systems,provides a mechanism to not only test for efficacy, but to quantify thesame by comparing the level of tdTomato to the luciferase andquantifying the same. Indeed, the ability to quantify the efficacy ofthe inhibition is performed by comparing the luciferase activity totdTomato activity, which is tumor selective and measures tumor size.

In preferred embodiments, the novel reporter system is a transducedmelanoma cell having transduced an EGFP-firefly luciferase fusion gene,under the control of E2F transcriptional response elements. Applicantstransduced mutant BRAF melanoma cells 1205Lu and mutant NRAS melanomacells, WM1366 to express EGFP-firefly luciferase fusion gene under thecontrol of E2F response elements. Accordingly, methods of the presentdisclosure take the transduced melanoma cell and apply an inhibitorycompound. The cells are then incubated for a period of time and thenfirefly luciferase activity is measured. The amount of inhibition of theCDK4/6 pathway can be determined by comparing the amount of fireflyluciferase expression to the amount of expression of tdTomato. Multiplegenotypically distinct melanoma models can be generated.

In this reporter system, hyper-phosphorylation of RB1 leads to theuncoupling of E2F and E2F-mediated induction of firefly luciferaseactivity. 1205Lu cells were chosen based on their ability to form tumorsin vivo and their utility in a previously developed ERK1/2 reportersystem. Tumor cells were also transduced with tdTomato fluorescentprotein to selectively monitor tumor growth. The tdTomato is expressedin every cell thus measurement of its activity measures the quantity ofcells present, wherein the Firefly luciferase activity measures theamount of E2F that is available to bind to its response elements in thereporter construct thus driving the signaling pathway. By comparing thetwo measurements, the reporter system enables one to quantify the amountof block of the compound of interest.

Accordingly, a method of the present disclosure involves utilizingmodified cells involving a modified E2F reporter system to monitor theefficacy of CDK4/6 inhibitor based therapies both in vitro and in vivo.Tumor cells are transduced with tdTomato fluorescent protein and anEGFP-firefly luciferase fusion gene under the control of E2Ftranscriptional response elements. In vitro, cells are thenappropriately treated with multiple compounds of interest and fireflyluciferase activity can be measured and normalized to the amount oftdTomato activity/actin levels in the cells. In vivo, reporter cells areinjected in immunodeficient nude mice and when the tumors arise, themice are treated with compounds of interest that show effectiveinhibition of luciferase activity in vitro. Firefly luciferase activitycan imaged and quantified and then normalized to tdTomato activity inthe tumors. By normalizing the amount of firefly luciferase activity totdTomato activity, the results of inhibition in the tumor can bequantified.

Therefore, an embodiment of the invention is the ability to quantify theamount of inhibition of a particular compound to inhibit reporteractivity in these cell models. To make this quantification, one comparesFirefly luciferase activity to tdTomato. The tdTomato fluorescence isaltered based on tumor size, whereas the firefly luciferase activity ismodified based on the inhibition of the particular compound on thesignaling pathway. Thus, effective inhibition of the pathway by aparticular compound can be visualized by quantifying luciferase activitynormalized to tdTomato fluorescence. Cells that are resistant to aparticular compound will maintain high firefly luciferase/pathwayactivity.

The reporter cells described herein can be any cancer cell type. Indeed,by using the embodiments described herein, different types of cancercells can be transduced with the E2F dependent reporter construct totest different clinically relevant compounds for block of the E2Fpathway. Therefore, a preferred embodiment is directed to a method forgenerating a reporter cell line, comprising identifying a cancer cell,transducing the cancer cell with tdTomato fluorescent protein tospecifically measure tumor size and an EGFP-firefly luciferase fusiongene under the control of E2F transcriptional response elements tomeasure pathway activity. Cells are then appropriately treated with acompound of interest, incubated, and imaged, and the amount of tdTomatocan be compared to the firefly luciferase activity therein in either invitro or in vivo screening methods. By comparing the amount of tdTomatoto the amount of firefly luciferase expression, the results ofinhibition can be quantified.

In certain embodiments, a kit may also be suitable comprising (1) E2Fresponse elements-EGFP-Firefly luciferase plasmid/lentivirus, and (2)tdTomato fluorescent protein plasmid/lentivirus. Wherein, the componentscan be utilized in conjunction with a cancer cell to generate a reportercell line of interest, and test compounds of interest under the methodsas described herein. In preferred embodiments, applicants' specificreporter system utilizes firefly luciferase. However, other suitableluciferase reporters e.g. Renilla luciferase or fluorescent proteinse.g. mCherry may be interchangeably utilized to optimize the sensitivityof the system for the particular cell type.

A further method is a method for screening compounds for inhibitoryproperties on E2F melanoma cells comprising applying two or morecompounds of interest to a modified reporter cell, comparing the amountof tdTomato expression to the amount of firefly luciferase expression inthe cell and quantifying the results of the inhibition of the compound.Therefore, a method for screening compounds of interest for inhibitoryproperties on E2F reporter melanoma cells, provides for the ability toexamine the efficacy of multiple compounds onto a cell at once.Furthermore, in a high-throughput screen, dozens of wells can beutilized at once to test one or more compounds per well, to increase therate of testing of compounds. These screens provide an opportunity toidentify compounds that show greatest activity to inhibit E2F pathwaysuch as upstream inhibitors of the E2F pathway e.g. inhibitors of theMAP Kinase pathway (MEK inhibitors and ERK inhibitors). Compounds thatare particularly effective in the screen can then be further tested inmouse xenograft models.

A further embodiment is directed to a method for screening compoundsusing an in vitro and in vivo screening process. The method comprises afirst in vitro screening process to identify compounds that have a blockof 50% or greater than control. These compounds meeting the limitationof 50% block can then be further tested in vivo, wherein the reportercells are injected into an immunodeficient nude mice and tumors areallowed to grow. When the tumors arise, the mice are treated andpathway/luciferase activity is imaged and quantified along with tdTomatofluorescent protein activity. Normalized firefly luciferase activity totdTomato activity can be quantified and represents the amount of pathwayinhibition when compared to control mice. The 50% block limitation canbe modified from a percent of about 10% to about 100%, in order to havemore or less restrictive screening process.

A further advantage of the system is as a method for testing inhibitorycompounds in vivo in the same tumor. This permits temporal analysis ofthe same tumor. Pathway activity can be plotted against time as the onetumor is monitored in the same mouse to either regress or acquireresistance.

A further advantage is the use of the reporter to determine effectivescheduling and dosing of compounds before the drug is moved to theclinic. Combination therapy can induce severe toxicities in patients.Utilizing the reporter system, drug concentrations can be optimized bytesting various drug concentrations and combinations that results in thehighest inhibition on pathway activity with the least present sideeffects/toxicities. Similarly, scheduling of drugs such as a three weekON and 1 week off schedule versus other schedules can also be tested bylooking at inhibition of pathway activity. Reactivation of the pathwayalso corresponds with an actively proliferating tumor and subsequentresistance to drugs.

The reporter construct may also be transfected into different cancercell lines as well as individual patient derived tumors. In patientderived xenograft reporter models, a tumor is extracted from a patient.The tumor is homogenized to single cell cultures and subsequently, theE2F reporter construct and tdTomato fluorescent protein is transfectedinto the tumor cells. After transfection the cells are xenografted intomice and the mice are treated with test compounds. Firefly luciferaseactivity normalized to tdTomato activity measures the efficacy of thetest compound with percent inhibition of E2F activity as the ouput.Patient derived xenografts are clinically relevant unlike establishedhuman cell lines as they are more heterogeneous and can allow forindividualized precision medicine.

Indeed, precision medical treatments may allow for an individual patientcancer cell to be systematically be tested with hundreds of potentialcompounds of interest before determining a precise individual compoundor cocktail combination of compounds that provides for the single besttreatment for the individual and their particular cancer cell/form.Because of the ability to test these quickly in mouse models as well asin in vitro models, such testing may allow for precision medicine andindividualized care.

EXAMPLES

The following non-limiting examples are provided to give examples of themanner and mechanism for creating the reporter cells, testing the cellsin vitro and in vivo and providing examples of methods for testing andusing the reporter cells described herein.

Applicants using the reporter system described herein tested anddetermined that concurrent targeting of CDK4/6 and MEK resulted inenhanced deleterious effects in cell viability and apoptosis in bothBRAF and NRAS mutant melanoma cells. Furthermore, mechanisticinvestigation uncovered one potential mediator of response to CDK4/6plus MEK inhibitors as survivin, a known survival factor in melanoma. Onthe basis of these findings, the in vitro results were corroborated todemonstrate significant tumor regressions in vivo with simultaneousCDK4/6 and MEK inhibition compared to single agents alone. The efficacyof the combination was demonstrated using the in vivo E2F activityreporter melanoma xenograft system to temporally quantitate the effectof the inhibitors and allow for the quantitative and temporal analysisof pathway reactivation during acquired resistance.

To determine the sensitivity of melanoma cells to CDK4/6 inhibition, theresponse of a genetically diverse panel of melanoma cell lines topalbociclib was examined (FIG. 3A). FIGS. 3A-3D depicts differentialresponse of melanoma cells to CDK4/6 inhibition. FIG. 3A depicts Westernblot of potential biomarkers of response to palbociclib in a panel ofmelanoma cell lines. FIG. 3B depicts sensitivity of melanoma cells topalbociclib. GI50 values were generated from dose-dependent curves fromMTT cell viability assays. Each bar represents the average of threeindependent experiments. FIG. 3C depicts palbociclib treated melanomacells were analyzed by Western blotting for RB1 phosphorylation andexpression of RB1 and cyclin A. FIG. 3D depicts that melanoma cells weretreated with palbociclib for 24 hours and analyzed by PI staining. Therelative distribution of cells in the subG1, G1, S and G2M phases of thecell cycle is shown. E. Mice bearing 1205Lu xenografts were treated withcontrol chow (n=4) or palbociclib chow (n=8). (Error bars represent SEM,*p<0.05).

As depicted in FIG. 3, cells were treated with increasing concentrationsof palbociclib in order to determine the GI50 for each cell line. Theresponse of melanoma cells to palbociclib was heterogeneous with thewild-type (WT) BRAF and NRAS cell lines, CHL-1 and BOWES, showing thehighest sensitivity (FIG. 3B). By contrast, the mutant BRAF and RB1 nullcell line, SKMEL207, showed the lowest sensitivity. No clear correlationwas observed between CDKN2A mutational status or INK4 family proteinexpression and the response to palbociclib within the panel (FIGS. 3Aand 3B). For example, SBcl2 cells are p16INK4A-deficient concurrent withhigh CDK4 and cyclin D1 expression but were relatively resistant toCDK4/6 inhibition (FIG. 3A, 3B). Phosphorylation of RB1 and cyclin A2expression were decreased within 24 hours of exposure to palbociclibwith effects more evident at low concentrations (0.05 μM) in the moresensitive cell lines.

Irrespective of the GI50, acute treatment with 0.5 μM palbociclibresulted in decreased phosphorylation/expression of RB1 and cyclin A2 inall cell lines except RB1-null, SKMEL207 (FIG. 3C). To furthercharacterize the cellular response to palbociclib as a single agent, apanel of cells was treated with palbociclib and performed propidiumiodide (PI)-based cell cycle analysis. Twenty-four hour treatment ofpalbociclib induced a G0/G1 cell cycle arrest but not cell deathrepresented by a lack of subG1 accumulation (FIG. 3D). Furthermore,palbociclib as a single agent initially suppressed 1205Lu tumor growthin vivo but progressive growth ensued (FIG. 3E). Taken together, theseresults show that the response of melanoma cells to palbociclib isheterogeneous, may not clearly stratify to any one genotype, and leadsto cytostatic effects but neither cell death nor tumor regression.

CDK4/6 and MEK Inhibitors Synergize to Inhibit the Growth of BRAF- andNRAS-Mutant Melanoma Cell Lines.

Due to the inability of single agent palbociclib to induce cell death,whether CDK4/6 targeting sensitized melanoma cells to MEK targeting isexplored. Treatment with the MEK inhibitor, trametinib, blocked thegrowth of all cell lines tested although RB1-deficient mutant BRAFSKMEL207 cells displayed decreased sensitivity. Since the CDK4/6 plusMEK inhibitor combination represents a targeted inhibitor option that isapplicable across all melanoma genotypes, cell lines were treated withpalbociclib alone, trametinib alone or both inhibitors in combinationover a fixed-ratio, 7-point concentration range for 96 hours. Thecombination of palbociclib and trametinib reduced the viability of BRAFand NRAS mutant cell lines compared to single agent treatments (FIG.4A).

FIG. 4 depicts enhanced effects of combined MEK and CDK4/6 inhibition inBRAF and NRAS mutant lines in vitro. FIG. 4A depicts MTT cell viabilityassays of WT/WT, BRAF and NRAS mutant lines treated for four days withsingle agent (trametinib or palbociclib) or a fixed-ratio (1:100)combination (trametinib plus palbociclib) of both compounds (error barsrepresent SD). FIG. 4B depicts Calcusyn combination indices at medianeffective dose (ED50) generated from MTT assays in FIG. 4A. FIG. 4Cdepicts melanoma cells were plated at low density, treated with DMSO,trametinib (10 nM), palbociclib (1 mM) or the combination. After 1 week,cultures were stained with crystal violet.

On the other hand, there was only a modest effect of the combinatorialtreatment on CHL-1 and BOWES cells (both WT BRAF/WT NRAS) and SKMEL207(FIG. 4A). Calcusyn analysis revealed a strong synergism between thedrug combination in mutant BRAF and mutant NRAS cells at medianeffective dose (ED50) but only moderate to slight synergism in CHL-1,BOWES and SKMEL207 cells (FIG. 4B). Effective long-term responses to thecombinatorial treatment in A375 and SBcl2 cells were also confirmed bycolony formation assay (FIG. 4C). Again, the palbociclib plus trametinibcombination was less effective in CHL-1 and SKMEL207 cells compared tothe other cell lines tested.

Down-Regulation of Survivin Associates with Response to CDK4/6 Plus MEKInhibitor Combination.

Therefore, a question arose as to whether the enhanced effect seen withthe combination of CDK4/6 and MEK inhibition was due to apoptosis. Anincreased annexin V staining was observed when CDK4/6 and MEK weresimultaneously inhibited in some cells (e.g. A375 and SBcl2) but notothers (FIG. 5A). In WM793 and 1205Lu cells, MEK inhibition alone wassufficient to induce apoptosis. Consistent with these data, trametinibincreased cleaved PARP levels in cell lines that showed enhanced effectsto the combination.

FIG. 5 depicts sensitivity to combined CDK4/6 and MEK inhibition isassociated with survivin depletion in sensitive cells. FIG. 5A. depictsa representative annexin V staining of human melanoma lines treated withtrametinib (5 nM) and palbociclib (0.5 μM) treatment alone or acombination of both compounds for 48 hours (n=3, error bars=SD fromtriplicate samples, *p<0.05). FIG. 5B depicts A375 cells were treatedwith single agent or combination of trametinib (5 nM) and palbociclib(0.5 μM) for 24 hours. DMSO represents the control. Lysates wereanalyzed by RPPA. The heatmap shows the most significantly regulatedproteins (p<0.01). FIG. 5C depicts that elevated levels of Bim-EL intrametinib and combo-treated lysates. FIG. 5D depicts that 1205Lu cellswere transduced with silencing RNA to Bim in the presence or absence oftrametinib (5 nM) and palbociclib (0.5 μM). Knockdown of Bim rescuedapoptotic phenotype elicited by trametinib and palbociclib treatments.FIG. 5E depicts fold change in BIRC5/survivin regulation after 24 hoursof treatment with indicated inhibitors. Two independent sets of testswere carried out and representative data is shown. FIG. 5F depictswestern blotting analysis of survivin in cell lines in resistant (CHL-1,Bowes, SKMEL207) and sensitive (A375, WM793, 1205Lu, SBcl2, WM1346,WM1366) cell lines in basal state as well as following treatment withtrametinib and/or palbociclib for 48 hours. FIG. 5G depicts NRAS mutantmelanoma tumor explant treated with DMSO, trametinib (50 nM),palbociclib (0.5 mM) or the combination.

To analyze signaling proteins that may be perturbed by treatment ofpalbociclib and/or trametinib, RPPA profiling was performed on A375cells treated with either single agent or with the combination.Phospho-RB1 and FOXM1, two established substrates of CDK4/6, werecooperatively repressed by co-inhibition of CDK4/6 and MEK. Thepro-apoptotic protein, Bim-EL, was up-regulated by trametinib treatmentbut unaffected by palbociclib. Effects on Bim-EL levels in A375 cellswas validated by Western blotting and also observed in combinationinhibitor sensitive BRAF and NRAS mutant lines (FIG. 5C). Moderatechanges were detected in Bim-EL levels in response to trametinib in thecombination-resistant cell lines, CHL-1, BOWES and SKMEL207 (FIG. 5C).Finally, the requirement of Bim induction for the apoptosis induced bycombination treatment in the sensitive, 1205Lu cell line, was tested.Knockdown of Bim in 1205Lu cells partially inhibited trametinib pluspalbociclib-induced apoptosis as detected by reduced annexin V staining(FIG. 5D).

Since Bim-EL was induced by MEK inhibition alone, the mechanistic basisof the drug synergy was explored by testing for modulation of apoptoticgenes on a quantitative PCR array. Among the 84 survival-related genes,BIRC5 exhibited the greatest change in levels in response to concurrentinhibition of MEK and CDK4/6 that was consistent between the two mostsensitive cell lines, A375 and SBcl2 (FIG. 5E). BIRC5 encodes forsurvivin, a protein belonging to the inhibitor of apoptosis (IAP)family. A similar trend in the modulation of protein levels of survivinby Western blot was validated. Concurrent treatment with trametinib andpalbociclib completely ablated the expression of survivin in sensitivemutant BRAF and mutant NRAS cell lines, which coincided with thedephosphorylation of RB1 (FIG. 5F). By contrast, the combination oftrametinib and palbociclib did not affect survivin levels in lesssensitive lines, CHL-1, BOWES, and SKMEL207 (FIG. 5F). Althoughexpression of the pro-survival protein, BCL2, was modestly affected onthe PCR array, there were no alterations at the protein level (FIG. 5F).

Finally, a primary human tumor explant culture was utilized tointerrogate whether the combinatorial treatment led to an enhancedapoptotic response. Ex vivo treatment of NRAS-mutant melanoma tissue for48 hours with single agents alone led to decreased survivin expressionthat was further suppressed in combination treated tumors (FIG. 5G).Bim-EL expression and cleaved PARP levels were also enhanced incombination-treated lysates. These data suggest that Bim-ELup-regulation and survivin down-regulation may be markers of theresponse to CDK4/6 and MEK inhibitor combinations.

Survivin is Essential for the Survival of Melanoma Cells.

To investigate whether survivin is required for survival, survivinexpression was silenced in a panel of melanoma cells (FIG. 6A). Indeed,FIG. 6 depicts that survivin is essential for the survival of melanomacells. FIG. 6A depicts melanoma cells were transfected with control orsurvivin-targeting siRNA #1 and #2 for 72 hours and lysates wereanalyzed by Western blot. FIG. 6B depicts knockdown of survivin leads todecreased cell viability in melanoma cells (n=4, error bars=SD *p<0.01).FIG. 6C depicts cell cycle analysis of melanoma cells after 72 hours oftransfection with two distinct siRNA specific for survivin. Bar graphswere generated from averages of three independent knockdown experiments.FIG. 6D depicts that melanoma cells were transfected with control or twodistinct survivin-targeting siRNA for 72 hours before they were analyzedfor annexin V staining by flow cytometry (n=3, error bars=SD, *p<0.05).FIG. 6E depicts that CHL-1 cells were transfected with control orsurvivin siRNA before they were treated with either DMSO or thecombination of trametinib (5 nM) plus palbociclib (0.5 mM) (n=3, errorbars=SD, *p<0.05 **p<0.01).

Knockdown of survivin led to a decrease in cell viability as compared tocontrol transfectants in MTT assays (FIG. 6B). In addition to its rolein resistance to apoptosis, mitotic properties of survivin have beendescribed; thus, it was examined whether depletion of survivin leads tocell cycle arrest in G2/M phase or cell death in melanoma cells by PIand annexin V staining. Survivin knockdown in a panel of melanoma cellsled to accumulation in the sub-G1 phase post-knockdown indicative ofapoptotic cell response but not G2-M accumulation after 72 hours (FIG.6C). Depletion of survivin at earlier time points (24 hours) did notinduce an arrest in G2-M (data not shown). Similarly by annexin Vstaining, survivin depletion induced significant apoptosis in themajority of the cell lines we tested across different genotypes (FIG.6D).

Next, it was determined whether knockdown of survivin was capable ofsensitizing combination-resistant CHL-1 cells to the CDK4/6-MEKinhibitor combination. Depletion of survivin in conjunction withcombination treatment increased cell death in CHL-1 cells (FIG. 6E).These data indicate that survivin depletion regulates the response ofmelanoma cells to the combination of CDK4/6 and MEK inhibitors.

Combined CDK4/6 and MEK Inhibition in vivo Potently Inhibits E2FReporter Activity and Regresses Melanomas.

Based on encouraging in vitro data suggesting the benefit of combinedCDK4/6 and MEK inhibition, the studies were extended in vivo. FIG. 7depicts that the combination of CDK4/6 and MEK inhibition is synergisticin vivo. FIG. 7A depicts the E2F reporter system for measuring theefficacy of CDK4/6 and MEK inhibitors in vivo. Firefly luciferase isexpressed under the control of E2F response elements. FIG. 7B depictsthat 1205LuTR reporter cells were treated with DMSO or palbociclib (0.5μM) for 48 hours (error bars=SD, *p<0.001, n=3). FIG. 7C depicts thatmice bearing 1205Lu xenografts were treated with control, MEK inhibitor(PD032901) alone, CDK4/6 inhibitor (palbociclib) alone, or incombination. Tumor volume was measured by digital caliper (errorbars=SEM, *p<0.001 comparing combo to single agents and control).CR=complete response. FIG. 7D depicts analysis of E2F reporter activitynormalized to tdTomato fluorescent protein activity in 1205Luxenografts. FIG. 7E depicts images from individual mice bearing 1205Luxenografts with tumor progression associated with high E2F reactivationin MEK inhibitor-treated mice and low E2F reactivation in CDK4/6inhibitor and combo-treated inhibitor mice. FIG. 7F depicts mice bearingWM1366 xenografts were treated with control, MEK inhibitor (PD032901),CDK4/6 inhibitor (palbociclib), or in combination. Tumor volume wasmeasured by digital caliper (error bars=SE, *p<0.001 comparing combo tosingle agents and control). CR=complete response. FIG. 7G depictsrepresentative images of 1205Lu xenografts taken from mice fed vehicle,MEK inhibitor, CDK4/6 inhibitor and combo-laced chow analyzed for Ki67.FIG. 7H depicts quantitation of Ki67 positive cells (% of total cells)taken from mice fed vehicle (n=2), MEK inhibitor (n=4), CDK4/6 inhibitor(n=4) and combo laced chow (n=4). Combo versus single regiments,*p<0.05, **p<0.001.

To test the inhibition in vivo, the E2F-dependent luciferase reportercell line was developed to enable temporal quantification of the effectsof CDK4/6 inhibitor based treatments in a quantitative, non-invasive,and tumor-selective manner. In this reporter system,hyper-phosphorylation of RB1 leads to the uncoupling of E2F andE2F-mediated induction of firefly luciferase activity (FIG. 7A). 1205Lucells were chosen based on their ability to form tumors in vivo andtheir utility in a previously developed ERK1/2 reporter system.

Tumor cells were also transduced with tdTomato fluorescent protein toselectively monitor tumor growth. In vitro, 1205Lu reporter cells showeda 60% reduction in firefly luciferase activity when normalized to actinlevels in the cells following 48 hours of treatment with palbociclib(FIG. 7B). Silencing RNA targeted to CDK4 and/or CDK6 also resultedin >50% decrease in firefly luciferase activity (FIG. 8). In FIG. 81205Lu reporter cells were transfected with silencing RNA to controlsequence, CDK4, CDK6 or both CDK4 and CDK6. After 72 hours oftransfection, whole cell lysates were collected and luciferase activitywas measured in each sample. Firefly luciferase activity was normalizedto actin levels in each sample.

This system can be used for a high throughput screening of potentialinhibitors prior to their utility in vivo. Those of ordinary skill inthe art will recognized that the reporter cells as described herein canbe utilized in one of many screening protocols using individual slidesand cells or in multi-welled plates to increase throughput. Mechanismsto quantify and visualize the cells in these individual plates are wellknown to those of skill in the art, in addition to the examples andmethods described herein.

In a preferred embodiment, a pre-determined number of cells can betransferred to a well and treated with a compound of interest and acarrier vehicle. A further well can be treated with just the drugvehicle to serve as a control, as is known to one of ordinary skill inthe art. Suitable replicates can be included, and/or several differentcompounds of interest can be tested at a time. The treated cells arethen incubated as described herein, typically for between 12-72 hoursand then luminescence is measured to determine the amount of pathwayactivity. Suitable luminometers is known to one of ordinary skill in theart, and is able to quantify luciferase activity for each well andtherefore calculate the efficacy of the compound as compared to thecontrol.

The benefit to a screening model in vitro is that it can be furtherverified by use of the same cells in vivo. Indeed, a hypothetical set of1000 compounds can be quickly and easily screened for efficacy and thesecompounds can be quantified based on the ability of the compound toblock CDK4/6-E2F pathway. As known to one of ordinary skill in the art,superior compounds can be re-tested to confirm efficacy in a further invitro test, or can be then further tested in in vivo models such as amouse xenograft model. These models will also test for efficacy of blockof E2F pathway, but also to confirm and test for resistance to the drug.Resistance to the compound of interest can be tested by monitoringfirefly luciferase activity and tdTomato activity in a temporal mannerand looking for an increased in the value of firefly luciferase activitynormalized to tdTomato fluorescence which indicates that the E2F pathwayhas been reactivated and thus, progression of the cell cycle.

Indeed, in a performed xenograft mouse model, 1205Lu reporter cells wereintradermally injected into immunodeficient nude mice. Tumors wereallowed to form before treatment with control chow, MEK inhibitor(PD0325901) chow, CDK4/6 inhibitor (palbociclib) chow or chow containingboth inhibitors. In the single agent and combination treatment cohorts,there was suppression of reporter activity that was more rapid in theCDK4/6 inhibitor-based treatments (FIGS. 7B and 7C). While MEK inhibitorinitially maintained tumor volumes at a static level and even led to onecomplete regression (mouse #6), dramatic tumor regrowth was detected bytreatment day 44 that was frequently preceded by strong E2F reactivation(FIGS. 7B and 7C). Tumors from mice treated with CDK4/6 inhibitor aloneshowed low but continuous growth with persistent inhibition of the E2Fpathway (FIGS. 7B and 7C). Levels of E2F activity also remained low intumors from mice treated with the combination of MEK plus CDK4/6inhibitor and this regimen was the only treatment to produce significanttumor regressions in 1205Lu xenografts (FIGS. 7B, 7C and 7D). Althoughcombining inhibitors can induce toxicities, the weight of the mice inall four cohorts was comparable from the starting time to the time oftermination.

Similar results on tumor growth were observed with a second reportermodel in an NRAS-mutant melanoma background (FIG. 7E). In contrast tothe 1205Lu model, the response of WM1366 tumors to single agenttreatment groups were heterogeneous. However, combination treatmentresulted in uniform tumor regressions and resulted in two out of sixcomplete responses (FIG. 7E). E2F activity was diminished in the tumor(mouse #7) that showed complete response to the combination treatment.Fluctuations in E2F activity occurred in one tumor that did notcompletely regress (mouse #1) and E2F reactivation also preceded theincrease in tumor volume/tdTomato activity in combination-resistanttumors (mouse #2 and #5).

Analyses of the harvested tumors from the 1205Lu reporter model revealeda statistically significant decrease in proliferating Ki67 positivecells in combination treated samples when compared to single agent andcontrol treated tumors (FIG. 7F, 7G). Together, these data show that anE2F reporter xenograft model may be used to quantitate drug targetinhibition and its association with tumor growth inhibition in vivo.Furthermore, the combination of a MEK inhibitor with palbociclibprovides an effective therapeutic strategy for BRAF and NRAS-mutantmelanomas.

E2F Reactivation and Rapid Tumor Regrowth from Combo Drug Withdrawal.

In the 1205Lu reporter model, five out of nine mice treated with thepalbociclib/MEK inhibitor combination achieved complete response, asdefined by undetectable caliper measurements (FIG. 9A); however, weaktdTomato signal (<0.8×10¹⁰ p/sec/cm²/sr) was detected in position of thetumors in all five mice. As depicted in FIG. 9, tumor regrowth followingMEK and CDK4/6 inhibitor withdrawal is identified. FIG. 9A depicts micebearing 1205Lu xenografts that showed complete response from thecombination therapy were removed from treatment at day 51 and monitoredfor durable response. Mice were retreated with combination regimen atday 67. FIG. 9B depicts modulation of E2F activity in combinationtreated mice following drug removal and retreatment. E2F reactivation ispredictive of tumors that would later acquire resistance to thecombination treatment. The tumor from mouse #5 exhibited necrosis by day129 and was excluded from luciferase and tdTomato analysis. FIG. 9Cdepicts RPPA analysis on resistant tumors. Combination resistant tumorsclustered with CDK inhibitor resistant tumors using unsupervisedhierarchical clustering. ComboR 8 is a regressing tumor that wasisolated during regression on treatment. MEKR 9/10 was least similar toany other groups. Thus, the E2F reporter melanoma cell line that can beused to screen multiple compounds of interest.

To determine whether the complete responses were durable, these fivemice were released from drug treatment (treatment day 51). Within oneweek off drug, a rapid reactivation of the E2F pathway and tumorregrowth were observed in all mice except one (mouse #1) (FIG. 9B).After two weeks (treatment day 67), mice were retreated with thepalbociclib/MEK inhibitor combination. The tumors re-responded rapidlyto the combination drug as evident by dramatic inhibition of pathwayactivity. Overall there was only one complete response with no palpabletumor, residual tdTomato signal or E2F activity (mouse #2). Fluctuationsin E2F activity was observed in the other four remaining mice followedby tumor regrowth. Reactivation of the pathway was evident andpredictive of tumors that would later acquire resistance to thecombination therapy (e.g. mouse #6). These data suggest that residualdisease remains after first-line treatment with the CDK4/6 and MEKinhibitor combination and that tumors that regrow from residual diseaseoff drug are likely to acquire resistance following retreatment.

Finally, RPPA data was utilized to identify proteins regulated by tumorresistance. However, it was unclear whether the combination resistantgroup could be stratified to any single agent resistant group. Todetermine resistance, all the samples were clustered based on a list ofsignificant antibodies to reduce noise from random differences betweengroups. This separated the tumors into two major distinct clusters (FIG.9C). Notably, combination resistant tumors that regrew from subsequentwithdrawal and retreatment of the inhibitors formed a clear cluster withCDK inhibitor resistant tumors. MEKR#9/#10 and Combo-R #8 formed theirown clusters, as well as the control samples. Combo-R #8 was a samplethat regressed on-treatment and was isolated during regression. Takentogether, these data suggest that combination resistant tumors weremolecularly similar to tumors that slowly progressed on palbociclib.

By taking melanoma cells modified to express an E2F reporter, theapplicants were able to test inhibitory compounds for effect on thetumor and therefore certain methods using the modified cells asdescribed herein, may be suitable for testing of inhibitory compounds.The applicants utilized several compounds, including palbociclib(PD0332991) a selective inhibitor of CDK4/6 that inhibits G1-Sprogression in vitro thus eliciting a cytostatic effect on tumors.Indeed, there reporter cell lines enable testing of the combined effectof multiple compounds on a reporter cell, or to test several individualcompounds under high-throughput screening methods.

All patents and publications cited herein are hereby fully incorporatedby reference in their entirety. The citation of any publication is forits disclosure prior to the filing date and should not be construed asan admission that such publication is prior art or that the presentinvention is not entitled to antedate such publication by virtue ofprior invention.

Having now fully described this invention, it will be understood tothose of ordinary skill in the art that the methods of the presentinvention can be carried out with a wide and equivalent range ofconditions, formulations, and other parameters without departing fromthe scope of the invention or any embodiments thereof.

What is claimed is:
 1. A method for determining the efficacy of acompound of interest to inhibit E2 Factor (E2F) activity comprising: a.applying a compound of interest to a tumor cell, wherein said tumor cellcomprises a modified E2F reporter system to monitor the efficacy ofCDK4/6 inhibitor-based therapies, wherein said tumor cells aretransduced with tdTomato fluorescent protein and an EGFP-fireflyluciferase fusion gene under the control of E2F transcriptional responseelements; and, wherein the tumor cell is a melanoma cell harboring aserine/threonine protein kinase B-RAF (BRAF), a melanoma cell harboringa neuroblastoma RAS viral oncogene homolog (NRAS) mutation, or amelanoma cell wild type for both BRAF and NRAS; b. determining theamount of tdTomato activity and determining the amount of fireflyluciferase activity in the tumor cell, and c. comparing the amount oftdTomato activity to the amount of firefly luciferase activity whereinthe tdTomato activity measures tumor size and firefly luciferaseactivity measures pathway activity in the tumor; and quantitate theinhibition of the E2F reporter activity through visualizing the activityof the tdTomato and firefly luciferase and comparing the fireflyluciferase activity normalized to tdTomato activity.
 2. The method ofclaim 1, wherein the method is performed in vitro.
 3. The method ofclaim 1, wherein the method is performed in vivo, further comprising thesteps of: d. injecting the tumor cell line expressing the reportersystem subcutaneously or intradermally into a mouse model, whereintumors are allowed to form; and e. injecting said mouseintraperitoneally with luciferin wherein said firefly luciferaseactivity can be imaged and quantitated using an in vivo imaging system(IVIS) machine.
 4. The method of claim 3 wherein tdTomato activity canbe imaged and quantitated using an IVIS machine; and wherein fireflyluciferase activity is normalized to tdTomato activity to quantifyexisting pathway activity.
 5. The method of claim 1 comprisingadministering a second compound of interest to the tumor cell.
 6. Themethod of claim 5 comprising administering a third compound of interestto the tumor cell.